Deciphering the Double-Edged Role of IFITM3 during SARS-CoV-2 Infection

Division Of Basic Sciences - Nci

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Abstract

We have successfully produced HIV-based and VSV-based pseudovirus bearing the spike protein of SARS-CoV-1 and SARS-CoV-2 variants and produced cell lines that are permissive to these pseudoviruses. We have developed a protocol for transiently transfecting human ACE2 (the receptor for SARS-CoV-1 and SARS-CoV-2) and TMPRSS2 (a protease that activates the fusion potential of SARS-CoV-1 and SARS-CoV-2 spike proteins) into HEK293T cells stably expressing human IFITM1, IFITM2, IFITM3, and mutants thereof. We then challenged these cells with the pseudoviruses and found that the human IFITM proteins inhibit both SARS-CoV-1- and SARS-CoV-2-mediated entry into cells, albeit to different extents. Whereas IFITM3 strongly inhibits SARS-CoV-1-mediated entry, it only slightly inhibits that driven by SARS-CoV-2. Furthermore, if target cells express TMPRSS2, the inhibitory effect of IFITM3 is negligible. These results suggest that viruses utilizing TMPRSS2 have decreased sensitivity to IFITM proteins, indicating that TMPRSS2 usage may alter the virus entry route into the cell and may be a means for the virus to evade intrinsic immune barriers. To complement our studies, we are collaborating with Jacob Yount at Ohio State University, where infections with replication-competent SARS-CoV-2 can be performed. The first chapter of our collaborative work was published in the EMBO Journal (Shi et al., EMBO J., 2021). Interestingly, we found that IFITM3 localized to endosomes restricts SARS-CoV-2 infection, while IFITM3 at the plasma membrane promotes SARS-CoV-2 infection. This finding demonstrated that SARS-CoV-2 may coopt IFITM3 at the cell surface for its own benefit, and provides yet another explanation for why SARS-CoV-2 exploits a cell entry pathway at the cell surface while SARS-CoV-1 does not. Currently, we are investigating the mechanisms by which IFITM3 promotes SARS-CoV-2 fusion at the plasma membrane, and we are assessing whether the recently evolved variants of concern, including Omicron, exhibit the same capacity to use IFITM3 for its own benefit. We will explore how the amphipathic helix and its cholesterol binding activity contribute to the promotion of SARS-CoV-2 infection by IFITM3.